New crystallographic methods will be developed to experimentally map and analyze electronic charge density and electrostatic potential distributions in proteins. Test cases for the development will be a series of allosteric insulin structures, already well-characterized by high-resolution X-ray diffraction. Experimentally, we shall employ the powerful technologies of macromolecular cryocrystallography with synchrotron X-ray sources and area detectors to measure diffraction data to atomic resolution. Computationally, we shall develop procedures to introduce experimental electron density parameters transferable to protein crystals at atomic resolution from a database of results from analyses of amino acid and oligopeptide crystals at sub-atomic resolution. The new methods to be developed will be able to provide answers to questions about the electrostatic, as distinct from geometric, atomic structure of proteins that present-day protein crystallographic methods cannot answer. Such questions include ambiguous states of ionization/protonation due to pK values being different in different molecular environments, ambiguous states of water-counterion structure at protein surfaces, and ambiguous protein- water hydrogen bonding donor-acceptor relationships. Also of great importance, the new methods will provide an experimental basis for calibrating the now theoretically derived electrostatic parameters used in molecular modelling and simulation calculations widely employed in many areas of structural biology. With respect to problems of insulin structural biochemistry, in particular, we expect our work will help resolve important questions of ill-defined electrostatic interactions in, for example, insulin-hexamer (GluB13)6 clusters, which according to various biophysical and biochemical evidence are probably key pieces in the mechanism of T6 T3R3 R6 insulin allosterism.